International Journal of Molecular Sciences
Article Deletion of Gb3 Synthase in Mice Resulted in the Attenuation of Bone Formation via Decrease in Osteoblasts
Kazunori Hamamura 1,* , Kosuke Hamajima 1,2, Shoyoku Yo 1,2, Yoshitaka Mishima 1,2, Koichi Furukawa 3 , Makoto Uchikawa 4, Yuji Kondo 5, Hironori Mori 1,2, Hisataka Kondo 1, Kenjiro Tanaka 1, Ken Miyazawa 2, Shigemi Goto 2 and Akifumi Togari 1
1 Department of Pharmacology, School of Dentistry, Aichi Gakuin University, Nagoya 464-8650, Japan; [email protected] (K.H.); [email protected] (S.Y.); [email protected] (Y.M.); [email protected] (H.M.); [email protected] (H.K.); [email protected] (K.T.); [email protected] (A.T.) 2 Department of Orthodontics, School of Dentistry, Aichi Gakuin University, Nagoya 464-8650, Japan; [email protected] (K.M.); [email protected] (S.G.) 3 Department of Biomedical Sciences, Chubu University College of Life and Health Sciences, Kasugai, Aichi 487-8501, Japan; [email protected] 4 Japanese Red Cross Tokyo Blood Center, Tokyo 162-8639, Japan; [email protected] 5 Department of Biochemistry II, Nagoya University Graduate School of Medicine, Nagoya 464-8650, Japan; [email protected] * Correspondence: [email protected]; Tel.: +81-52-757-6743; Fax: +81-52-752-5988
Received: 27 August 2019; Accepted: 17 September 2019; Published: 18 September 2019
Abstract: Glycosphingolipids are known to play a role in developing and maintaining the integrity of various organs and tissues. Among glycosphingolipids, there are several reports on the involvement of gangliosides in bone metabolism. However, there have been no reports on the presence or absence of expression of globo-series glycosphingolipids in osteoblasts and osteoclasts, and the involvement of their glycosphingolipids in bone metabolism. In the present study, we investigated the presence or absence of globo-series glycosphingolipids such as Gb3 (globotriaosylceramide), Gb4 (globoside), and Gb5 (galactosyl globoside) in osteoblasts and osteoclasts, and the effects of genetic deletion of Gb3 synthase, which initiates the synthesis of globo-series glycosphingolipids on bone metabolism. Among Gb3, Gb4, and Gb5, only Gb4 was expressed in osteoblasts. However, these glycosphingolipids were not expressed in pre-osteoclasts and osteoclasts. Three-dimensional micro-computed tomography (3D-µCT) analysis revealed that femoral cancellous bone mass in Gb3 synthase-knockout (Gb3S KO) mice was lower than that in wild type (WT) mice. Calcein double labeling also revealed that bone formation in Gb3S KO mice was significantly lower than that in WT mice. Consistent with these results, the deficiency of Gb3 synthase in mice decreased the number of osteoblasts on the bone surface, and suppressed mRNA levels of osteogenic differentiation markers. On the other hand, osteoclast numbers on the bone surface and mRNA levels of osteoclast differentiation markers in Gb3S KO mice did not differ from WT mice. This study demonstrated that deletion of Gb3 synthase in mice decreases bone mass via attenuation of bone formation.
Keywords: glycosphingolipids; globoside (Gb4); osteoblasts; bone metabolism
1. Introduction Glycosphingolipids are considered to play an important role in the development and maintenance of various organs and tissues [1–3]. These have also been shown to be critical for protection against
Int. J. Mol. Sci. 2019, 20, 4619; doi:10.3390/ijms20184619 www.mdpi.com/journal/ijms Int. J. Mol. Sci. 2019, 20, 4619 2 of 13
Int. J. Mol. Sci. 2019, 20, x 2 of 13 inflammationGlycosphingolipids and degeneration are considered [4]. Glycosphingolipids to play an important are divided role in into the three development main series, and i.e., ganglioside-series,maintenance of globo-series, various organs and and lacto tissues/neolacto-series. [1–3]. These Ofhave these, also gangliosides been shown haveto be beencritical reported for to beprotection involved inagainst bone inflammation metabolism and [5– 9degeneration]. For instance, [4]. Glycosphingolipids it was reported thatare divided a-series into ganglioside three GD1amain and GM1series, promote i.e., ganglioside osteoblastic‐series, di ffgloboerentiation‐series, and of bone lacto/neolacto marrow‐ mesenchymalseries. Of these, stem gangliosides cells (MSCs) and tendonhave been stem reported cells [to5– be8]. involved Recently, in bone we demonstrated metabolism [5–9]. that For a instance, lack of GD3it was synthase, reported that which a‐ is responsibleseries ganglioside for the generation GD1a and of GM1 all b-seriespromote osteoblastic gangliosides, differentiation in mice results of bone in marrow the attenuation mesenchymal of bone stem cells (MSCs) and tendon stem cells [5–8]. Recently, we demonstrated that a lack of GD3 synthase, loss with aging [9]. However, there have been no reports on the presence or absence of expression of which is responsible for the generation of all b‐series gangliosides, in mice results in the attenuation globo-series glycosphingolipids in osteoblasts and osteoclasts, and the roles of these glycosphingolipids of bone loss with aging [9]. However, there have been no reports on the presence or absence of in boneexpression metabolism. of globo‐series glycosphingolipids in osteoblasts and osteoclasts, and the roles of these Globo-seriesglycosphingolipids glycosphingolipids in bone metabolism. have a common core structure, Galα1,4-Galβ1,4-Glc-ceramide (globotriaosylceramide;Globo‐series glycosphingolipids Gb3), which is have synthesized a common by coreα 1,4-galactosyltransferasestructure, Galα1,4‐Galβ1,4‐Glc (Gb3‐ceramide synthase A4galt(globotriaosylceramide;) from lactosylceramide Gb3), (LacCer) which (Figure is synthesized1). Globo-series by α1,4‐ glycosphingolipidsgalactosyltransferase such(Gb3 assynthase Gb3, Gb4 (globoside),A4galt) andfrom Gb5 lactosylceramide (galactosyl (LacCer) globoside) (Figure have 1). not Globo only‐series been glycosphingolipids used as markers such for as stem Gb3, cells Gb4 and tumors(globoside), but are also and considered Gb5 (galactosyl to regulate globoside) immune have system not only and been maintain used as stemness markers [ 10for– 13stem]. Gb3 cells has and been characterizedtumors but as theare Palsok antigen considered on red to regulate blood cells immune [14] and system is considered and maintain to bestemness a tumor [10–13]. antigen Gb3 of has Burkitt been characterized as the Pk antigen on red blood cells [14] and is considered to be a tumor antigen lymphoma [10]. This glycosphingolipid has also been recognized as a receptor for verotoxins secreted of Burkitt lymphoma [10]. This glycosphingolipid has also been recognized as a receptor for by pathogenic Escherichia coli O157 [15]. Gb4 is a major component among glycosphingolipids in human verotoxins secreted by pathogenic Escherichia coli O157 [15]. Gb4 is a major component among erythrocytesglycosphingolipids [16] and is anin human essential erythrocytes structure [16] for bloodand is an group essential P antigen structure [17 for]. Gb4 blood was group also P reportedantigen to be an[17]. endogenous Gb4 was ligand also forreported Toll-like to be receptor an endogenous 4-myeloid ligand differentiation for Toll‐like factor receptor 2 (TLR4-MD-2) 4‐myeloid and attenuatesdifferentiation lipopolysaccharide factor 2 (TLR4 (LPS)‐MD toxicity‐2) and [ 13attenuates]. Gb5, whichlipopolysaccharide is also named (LPS) stage-specific toxicity [13]. embryonic Gb5, antigen-3which (SSEA-3), is also isnamed expressed stage‐specific at an early embryonic developmental antigen‐3 stage(SSEA and‐3), isis usedexpressed as a marker at an early for stem cells [11developmental]. stage and is used as a marker for stem cells [11].
LacCer Gb3 Gb3 synthase cer cer
Gb4 synthase Gb4 (globoside) Gb5 Gb5 (SSEA-3) synthase cer cer
cer : ceramide : Glucose : Galactose : N-acetylgalactosamine
Figure 1. Synthetic pathway of globo-series glycosphingolipids. Figure 1. Synthetic pathway of globo‐series glycosphingolipids.
In theIn present the present study, study, we we used used flow flow cytometry cytometry to to examine examine the the expression expression levels levels of ofglobo globo-series‐series glycosphingolipidsglycosphingolipids (Gb3, (Gb3, Gb4, Gb4, and and Gb5) Gb5) in in MC3T3 MC3T3 E1E1 mousemouse osteoblast osteoblast-like‐like cells, cells, SaM SaM-1‐1 human human osteoblastosteoblast cells, cells, RAW264.7 RAW264.7 mouse mouse pre-osteoclasts, pre‐osteoclasts, and and primary primary cultured cultured pre pre-osteoclasts‐osteoclasts derived derived from from mousemouse bone bone marrow marrow cells. cells. To evaluateTo evaluate the the involvement involvement ofof globo-seriesglobo‐series glycosphingolipids glycosphingolipids in bone in bone metabolismmetabolismin vivo in, vivo, femoral femoral cancellous cancellous bone bone mass mass in wild in wild type type (WT) (WT) and and Gb3 Gb3 synthase-knockout synthase‐knockout (Gb3S KO) mice(Gb3S were KO) analyzed mice were using analyzed three-dimensional using three‐dimensional micro-computed micro‐ tomographycomputed tomography (3D-µCT). (3D Furthermore,‐μCT). we conductedFurthermore, calcein we double conducted labeling calcein to examine double thelabeling roles ofto globo-series examine the glycosphingolipids roles of globo‐series in bone glycosphingolipids in bone formation. We employed bone histomorphometric analyses using formation. We employed bone histomorphometric analyses using hematoxylin and eosin (HE) and hematoxylin and eosin (HE) and tartrate‐resistant acid phosphatase (TRAP) staining. We also tartrate-resistant acid phosphatase (TRAP) staining. We also examined the expression levels of examined the expression levels of differentiation markers of osteoblasts and osteoclasts in long bones differentiation(femur and markers tibia) from of osteoblastsWT and Gb3S and KO osteoclastsmice using quantitative in long bones real‐ (femurtime PCR. and tibia) from WT and Gb3S KO miceWe report using here quantitative that deletion real-time of Gb3 PCR.synthase in mice results in significant attenuation of bone Weformation, report hereleading that to deletiondecreased of bone Gb3 mass. synthase This study in mice is the results first to in report significant that Gb4 attenuation is expressed of in bone formation,osteoblasts, leading and to also decreased that globo bone‐series mass. glycosphingolipids This study is are the involved first to report in bone that metabolism. Gb4 is expressed in osteoblasts, and also that globo-series glycosphingolipids are involved in bone metabolism. Int. J. Mol. Sci. 2019, 20, 4619 3 of 13
Int. J. Mol. Sci. 2019, 20, x 3 of 13 2. Results 2. Results 2.1. Expression of Globo-Series Glycosphingolipids (Gb3, Gb4, and Gb5), and Gb3 and Gb4 Synthase Genes in Osteoblasts2.1. Expression of Globo‐Series Glycosphingolipids (Gb3, Gb4, and Gb5), and Gb3 and Gb4 Synthase Genes in Osteoblasts Expression levels of globo-series glycosphingolipids (Gb3, Gb4, and Gb5) in osteoblasts were analyzedExpression by flow cytometry levels of globo (Figure‐series2A,B). glycosphingolipids Both MC3T3 E1(Gb3, mouse Gb4, and osteoblast-like Gb5) in osteoblasts cells and were SaM-1 humananalyzed osteoblast by flow cells cytometry expressed (Figure Gb4 strongly, 2A,B). Both while MC3T3 Gb3 wasE1 mouse below osteoblast the limit‐like of detection cells and inSaM both‐1 cells human osteoblast cells expressed Gb4 strongly, while Gb3 was below the limit of detection in both (Figure2A,B). Gb5 in MC3T3 E1 cells was not detected and it was very weakly expressed in SaM-1. The cells (Figure 2A,B). Gb5 in MC3T3 E1 cells was not detected and it was very weakly expressed in expressionSaM‐1. ofThe Gb4 expression in MC3T3 of Gb4 E1 in cells MC3T3 was E1 decreased cells was after decreased induction after induction of osteoblastogenesis of osteoblastogenesis (Figure 2A). Consistent(Figure with2A). Consistent this result, with the expressionthis result, the levels expression of Gb3 levels (A4galt of) Gb3 and ( Gb4A4galt (B3galnt1) and Gb4) synthase (B3galnt1) genes weresynthase downregulated genes were by downregulated induction of osteoblastogenesis by induction of osteoblastogenesis (Figure2C). (Figure 2C).
FigureFigure 2. Expression 2. Expression of of glycosphingolipids glycosphingolipids (Gb3, Gb4, Gb4, and and Gb5), Gb5), and and Gb3 Gb3 and and Gb4 Gb4 synthase synthase genes genes in in osteoblasts.osteoblasts. (A) ( ExpressionA) Expression of of globo-series globo‐series glycosphingolipidsglycosphingolipids (Gb3, (Gb3, Gb4, Gb4, and and Gb5) Gb5) in MC3T3 in MC3T3 E1 cells E1 cells on dayson days 0, 7, 0, and 7, and 21 after 21 after induction induction of of osteoblastogenesis osteoblastogenesis byby flowflow cytometric analysis. analysis. (B ()B Expression) Expression of globo-seriesof globo‐ glycosphingolipidsseries glycosphingolipids (Gb3, (Gb3, Gb4, Gb4, and and Gb5) Gb5) in SaM-1in SaM‐ cells1 cells by by flow flow cytometric cytometric analysis. Red Red line: anti‐globo‐series glycosphingolipids (Gb3, Gb4, and Gb5) monoclonal antibodies (mAbs) line: anti-globo-series glycosphingolipids (Gb3, Gb4, and Gb5) monoclonal antibodies (mAbs) (+), gray (+), gray line: anti‐globo‐series glycosphingolipids mAbs (‐). (C) mRNA expression of A4galt and line: anti-globo-series glycosphingolipids mAbs (-). (C) mRNA expression of A4galt and B3galnt1 in MC3T3 E1 cells on days 0, 7, and 21 after induction of osteoblastogenesis (n = 3). Data are expressed as mean S.D. The double asterisks indicate p < 0.01. ± Int. J. Mol. Sci. 2019, 20, 4619 4 of 13 Int. J. Mol. Sci. 2019, 20, x 4 of 13
2.2. No ExpressionB3galnt1 of Globo-Seriesin MC3T3 E1 cells Glycosphingolipids on days 0, 7, and 21 after (Gb3, induction Gb4, of and osteoblastogenesis Gb5), and Gb3 (n = 3). and Data Gb4 are Synthase Genes in RAW264.7 Cellsexpressed and as Primary mean ± S.D. Cultured The double Pre-Osteoclasts asterisks indicate p < 0.01. Gb3,2.2. Gb4, No and Expression Gb5 wereof Globo not‐Series detected Glycosphingolipids in RAW264.7 (Gb3, Gb4, cells and before Gb5), (Dayand Gb3 0) and and Gb4 after Synthase (Day 3) induction Genes in RAW264.7 Cells and Primary Cultured Pre‐Osteoclasts to osteoclasts (Figure3A). These glycosphingolipids were hardly expressed in primary cultured pre-osteoclastsGb3, derived Gb4, and from Gb5 mousewere not bone detected marrow in RAW264.7 cells before cells before (Day (Day 0) and 0) and after after (Day (Day 1)3) induction induction to osteoclasts (Figure 3A). These glycosphingolipids were hardly expressed in primary to osteoclastscultured (Figure pre‐osteoclasts3B). Consistent derived from with mouse the resultsbone marrow of the cells flow before cytometry, (Day 0) and the after expression (Day 1) levels of Gb3 (A4galtinduction) and to osteoclasts Gb4 (B3galnt1 (Figure) 3B). synthase Consistent genes with the were results at theof the limit flow ofcytometry, detection the expression in RAW264.7 cells before (Daylevels 0) of and Gb3 after (A4galt induction) and Gb4 ( (DayB3galnt1 2) and synthase 4) to genes osteoclasts were at the (Figure limit of3 C).detection Of note, in RAW264.7 mRNA levels of osteoclastcells diff erentiationbefore (Day 0) markers and after induction such as (Day nuclear 2 and factor 4) to osteoclasts of activated (Figure T-cells, 3C). Of cytoplasmic note, mRNA levels 1 (Nfatc1 ) and of osteoclast differentiation markers such as nuclear factor of activated T‐cells, cytoplasmic 1 (Nfatc1) tartrate-resistantand tartrate acid‐resistant phosphatase acid phosphatase (Trap) were (Trap upregulated) were upregulated in the in presence the presence of the of the receptor receptor activator of nuclear factoractivator kappa-B of nuclear ligand factor (RANKL) kappa‐B ligand (Figure (RANKL)3D). (Figure 3D).
Figure 3. FigureExpression 3. Expression of globo-series of globo‐series glycosphingolipids glycosphingolipids (Gb3, (Gb3, Gb4, Gb4, and Gb5), and Gb3 Gb5), synthase Gb3 synthasegene gene (A4galt) and(A4galt Gb4) and synthase Gb4 synthase gene gene (B3galnt1 (B3galnt1)) in RAW264.7 RAW264.7 cells cells and primary and primary cultured culturedpre‐osteoclasts pre-osteoclasts in in the presencethe presence or absence or absence of of RANKL. RANKL. (A (A) Expression) Expression of globo of‐ globo-seriesseries glycosphingolipids glycosphingolipids (Gb3, Gb4, and (Gb3, Gb4, and Gb5) in RAW264.7 cells on days 0 and 3 after administration of RANKL by flow cytometric analysis. (B) Expression of globo-series glycosphingolipids (Gb3, Gb4, and Gb5) in primary cultured pre-osteoclasts on days 0 and 1 after administration of RANKL by flow cytometric analysis. Red line: anti-globo-series glycosphingolipids (Gb3, Gb4, and Gb5) monoclonal antibodies (mAbs) (+), gray line: anti-globo-series glycosphingolipids mAbs (-). (C) mRNA expression of A4galt and B3galnt1 in MC3T3 E1 and RAW264.7 cells on days 0, 2, and 4 after administration of RANKL (n = 3). (D) mRNA expression of Nfatc1 and Trap in RAW264.7 cells on days 0, 2, and 4 after administration of RANKL (n = 3). Data are expressed as mean S.D. The single and double asterisks indicate p < 0.05 and ± p < 0.01, respectively. Int. J. Mol. Sci. 2019, 20, x 5 of 13
Gb5) in RAW264.7 cells on days 0 and 3 after administration of RANKL by flow cytometric analysis. (B) Expression of globo‐series glycosphingolipids (Gb3, Gb4, and Gb5) in primary cultured pre‐ osteoclasts on days 0 and 1 after administration of RANKL by flow cytometric analysis. Red line: anti‐ globo‐series glycosphingolipids (Gb3, Gb4, and Gb5) monoclonal antibodies (mAbs) (+), gray line: anti‐globo‐series glycosphingolipids mAbs (‐). (C) mRNA expression of A4galt and B3galnt1 in MC3T3 E1 and RAW264.7 cells on days 0, 2, and 4 after administration of RANKL (n = 3). (D) mRNA expression of Nfatc1 and Trap in RAW264.7 cells on days 0, 2, and 4 after administration of RANKL Int. J.( Mol.n = 3). Sci. Data2019, are20, 4619expressed as mean ± S.D. The single and double asterisks indicate p < 0.05 and p <5 of 13 0.01, respectively.
2.3.2.3. Decrease Decrease in in Femoral Femoral Cancellous Cancellous Bone Bone Mass Mass in in Gb3 Gb3 Synthase Synthase-Knockout‐Knockout (Gb3S (Gb3S KO) Mice BoneBone volume/total volume/total volume volume (BV/TV) (BV/TV) and and trabecular trabecular number number (Tb.N) (Tb.N) in in Gb3S Gb3S KO KO mice mice were significantlysignificantly lowerlower than than those those in WT in miceWT (Figuremice (Figure4A,C). Trabecular 4A,C). Trabecular separation separation (Tb.Sp) was (Tb.Sp) significantly was significantlyhigher than thathigher in WT than mice that (Figure in WT4D). mice Trabecular (Figure thickness 4D). Trabecular (Tb.Th) was thickness not significantly (Tb.Th) diwasfferent not significantlybetween WT different and Gb3S between KO mice WT (Figure and Gb3S4B). TheKO mice 3D- µ(FigureCT analysis 4B). The revealed 3D‐μCT that analysis deletion revealed of Gb3 thatsynthase deletion in mice of Gb3 results synthase in the in attenuation mice results of in bone the mass.attenuation of bone mass.
WT Gb3S KO A B 30 100 m)
20 * 50 10 Tb.Th ( BV/TV (%) BV/TV
0 0
C D
2 400 *
1.5 m) 300
** 1 200
0.5 Tb.Sp ( 100 Tb.N (1/mm) 0 0 FigureFigure 4.4. AttenuationAttenuation of of femoral femoral cancellous cancellous bone massbone inmass Gb3S in KO Gb3S mice. KO Analysis mice. of Analysis bone densitometry of bone µ densitometryof the distal region of the of distal the femur region in of WT the and femur Gb3S in KOWT miceand Gb3S by 3D- KOCT. mice Fifteen by 3D week-old‐μCT. Fifteen mice (male,week‐oldn = mice12 for (male, WT, n n= =11 12 for for Gb3S WT, KO). n = (11A) for Bone Gb3S volume KO)./total (A) volumeBone volume/total (BV/TV, %). volume (B) Trabecular (BV/TV, thickness %). (B) µ µ Trabecular(Tb.Th, m). thickness (C) Trabecular (Tb.Th, number μm). (C (Tb.N,) Trabecular 1/mm). number (D) Trabecular (Tb.N, 1/mm). separation (D) Trabecular (Tb.Sp, m). separation Data are expressed as mean S.D. The single and double asterisks indicate p < 0.05 and p < 0.01, respectively. (Tb.Sp, μm). Data are± expressed as mean ± S.D. The single and double asterisks indicate p < 0.05 and 2.4. Decreasep < 0.01, respectively. in Bone Formation Parameters by Gb3 Synthase Deficiency
2.4. DecreaseTo investigate in Bone whether Formation Gb3 Parameters synthase by regulates Gb3 Synthase bone formation, Deficiency we measured bone formation rate using calcein double labeling. The mineral surface/bone surface (MS/BS), mineral apposition rates To investigate whether Gb3 synthase regulates bone formation, we measured bone formation (MAR), and bone formation rate (BFR) were significantly lower in Gb3S KO mice than in WT mice rate using calcein double labeling. The mineral surface/bone surface (MS/BS), mineral apposition (FigureInt.5 J.). Mol. Sci. 2019, 20, x 6 of 13 rates (MAR), and bone formation rate (BFR) were significantly lower in Gb3S KO mice than in WT mice (Figure 5). AB
60 4
3 40 * m / / day) m
2 ** 20 1 MS/BS (%) MS/BS MAR ( 0 0
C WT Gb3S KO
/ day) 2 2 m / 3 1 m **
BFR ( BFR 0 FigureFigure 5. Attenuation 5. Attenuation of boneof bone formation formation by by Gb3 Gb3 synthasesynthase deficiency deficiency in in 15 15-week-old‐week‐old mice mice (male, (male, n = 6n = 6 for WT,for nWT,= 6 n for = 6 Gb3Sfor Gb3S KO). KO). (A ()A Mineral) Mineral surface surface/bone/bone surface (MS/BS, (MS/BS, %). %). (B) ( BMineral) Mineral apposition apposition rates rates (MAR,(MAR,µm/day). μm/day). (C) Bone(C) Bone formation formation rate rate (BFR, (BFR,µ μmm33// μµm2/day)./day). Data Data are are expressed expressed as mean as mean ± S.D. S.D.The The ± singlesingle and doubleand double asterisks asterisks indicate indicatep
2.5. Decrease in Osteoblast Number in Femoral Cancellous Bone and Osteogenic Differentiation Marker’s Gene Expression (Runt‐Related Transcription Factor 2, Alkaline Phosphatase, and Osteocalcin) in Long Bones by Gb3 Synthase Deficiency Osteoblast numbers/bone surface (Ob.N/BS) was significantly lower in Gb3S KO mice than in WT mice (Figure 6A,B). Furthermore, mRNA levels of runt‐related transcription factor 2 (Runx2), alkaline phosphatase (Alp), and osteocalcin were suppressed in Gb3S KO mice (Figure 7).
Figure 6. Decrease in osteoblast numbers by Gb3 synthase deficiency in 15‐week‐old mice (male, n = 10 for WT, n = 10 for Gb3S KO). (A) Images of hematoxylin and eosin (HE) staining of femoral cancellous bone. Results for WT mice (left) and Gb3S KO mice (right) are shown. The scale bars are 100 μm. Yellow arrow heads indicate osteoblasts. (B) Osteoblast numbers/bone surface (Ob.N/BS, number/mm). Data are expressed as mean ± S.D. The double asterisks indicate p < 0.01. Int. J. Mol. Sci. 2019, 20, x 6 of 13
AB
60 4
3 40 * m / / day) m
2 ** 20 1 MS/BS (%) MS/BS MAR ( 0 0
C WT Gb3S KO
/ day) 2 2 m / 3 1 m **
BFR ( BFR 0 Figure 5. Attenuation of bone formation by Gb3 synthase deficiency in 15‐week‐old mice (male, n = 6 for WT, n = 6 for Gb3S KO). (A) Mineral surface/bone surface (MS/BS, %). (B) Mineral apposition rates (MAR, μm/day). (C) Bone formation rate (BFR, μm3/ μm2/day). Data are expressed as mean ± S.D. The Int. J. Mol.single Sci. and2019 double, 20, 4619 asterisks indicate p < 0.05 and p < 0.01, respectively. 6 of 13
2.5. Decrease in Osteoblast Number in Femoral Cancellous Bone and Osteogenic Differentiation Marker’s 2.5. Decrease in Osteoblast Number in Femoral Cancellous Bone and Osteogenic Differentiation Marker’s Gene ExpressionGene Expression (Runt-Related (Runt‐Related Transcription Transcription Factor 2,Factor Alkaline 2, Alkaline Phosphatase, Phosphatase, and Osteocalcin) and Osteocalcin) in Long Bonesin Long by Gb3 SynthaseBones by Gb3 Deficiency Synthase Deficiency Osteoblast numbersnumbers/bone/bone surface surface (Ob.N (Ob.N/BS)/BS) was was significantly significantly lower lower in Gb3Sin Gb3S KO KO mice mice than than in WT in miceWT mice (Figure (Figure6A,B). 6A,B). Furthermore, Furthermore, mRNA mRNA levels oflevels runt-related of runt‐ transcriptionrelated transcription factor 2 (factorRunx2 2), ( alkalineRunx2), phosphatasealkaline phosphatase (Alp), and (Alp osteocalcin), and osteocalcin were suppressed were suppressed in Gb3S KOin Gb3S mice KO (Figure mice7). (Figure 7).
Figure 6. Decrease inin osteoblastosteoblast numbers numbers by by Gb3 Gb3 synthase synthase deficiency deficiency in in 15-week-old 15‐week‐old mice mice (male, (male,n = n10 = for10 for WT, WT,n = 10n = for 10 Gb3S for Gb3S KO). (AKO).) Images (A) Images of hematoxylin of hematoxylin and eosin and (HE) eosin staining (HE) of staining femoral of cancellous femoral bone.cancellous Results bone. for Results WT mice for (left)WT mice and Gb3S(left) and KO Gb3S mice (right)KO mice are (right) shown. are The shown. scale The bars scale are bars 100 µarem. Yellow100 μm. arrow Yellow heads arrow indicate heads osteoblasts. indicate osteoblasts. (B) Osteoblast (B) numbers Osteoblast/bone numbers/bone surface (Ob.N surface/BS, number (Ob.N/BS,/mm). Int. J. Mol. Sci. 2019, 20, x 7 of 13 Datanumber/mm). are expressed Data asare mean expressedS.D. as The mean double ± S.D. asterisks The double indicate asterisksp < 0.01. indicate p < 0.01. ± WT Gb3S KO 1.5 2 1.5
1.5 ) * 1 * 1 ) ) *
Alp 1 ( Runx2 ( 0.5
osteocalcin 0.5 0.5 ( Relative mRNA levels Relative mRNA levels Relative mRNA levels 0 0 0 FigureFigure 7.7. Suppression of gene expression of osteogenicosteogenic didifferentiationfferentiation markersmarkers ((Runx2Runx2,, AlpAlp,, andand osteocalcin)osteocalcin) by by Gb3 Gb3 synthase synthase deficiency. deficiency. RNA RNA isolated isolated from from femur femur and tibiaand intibia 15-week-old in 15‐week mice‐old (male, mice n(male,= 12 forn = WT, 12 forn = WT,12 for n = Gb3S 12 for KO). Gb3S Data KO). are Data expressed are expressed as mean asS.D. mean The ± singleS.D. The asterisk single indicates asterisk ± pindicates< 0.05. p < 0.05. 2.6. No Differences in Osteoclast Number and Expression of Osteoclast Differentiation Marker’s Genes (Nuclear 2.6. No Differences in Osteoclast Number and Expression of Osteoclast Differentiation Marker’s Genes Factor of Activated T-Cells, Cytoplasmic 1 and Tartrate-Resistant Acid Phosphatase) between WT and Gb3S KO Mice(Nuclear Factor of Activated T‐Cells, Cytoplasmic 1 and Tartrate‐Resistant Acid Phosphatase) between WT and Gb3S KO Mice Bone resorption parameters such as osteoclast surface/bone surface (Oc.S/BS) and osteoclast numbersBone/bone resorption surface parameters (Oc.N/BS) were such almost as osteoclast equivalent surface/bone in the WT andsurface Gb3S (Oc.S/BS) KO mice and (Figure osteoclast8A,B). Furthermore,numbers/bone nosurface differences (Oc.N/BS) in were nuclear almost factor equivalent of activated in the T-cells,WT and cytoplasmicGb3S KO mice 1 (Figure (Nfatc1 8A,B).) and tartrate-resistantFurthermore, no differences acid phosphatase in nuclear (Trap factor) mRNA of activated levels T were‐cells, found cytoplasmic between 1 (Nfatc1 WT and) and Gb3S tartrate KO‐ miceresistant (Figure acid8 C).phosphatase (Trap) mRNA levels were found between WT and Gb3S KO mice (Figure 8C).
WT Gb3S KO A B
30 15
20 10
10 5 Oc.S/BS(%)
0 Oc.N/BS(N/mm) 0
C 1.5 1.5
1 1 ） ） Trap
0.5 （ 0.5 Nfatc1 （ Relative mRNA levels 0 Relative mRNA levels 0 Figure 8. No effects of Gb3 synthase deficiency on bone resorption parameters and gene expression of osteoclast differentiation markers (Nfatc1 and Trap). (A) Osteoclast surface/bone surface (Oc.S/BS, %) in 15‐week‐old mice (male, n = 9 for WT, n = 10 for Gb3S KO). (B) Osteoclast numbers/bone surface (Oc.N/BS, number/mm) in 15‐week‐old mice (male, n = 9 for WT, n = 10 for Gb3S KO). (C) mRNA expression of Nfatc1 and Trap. RNA isolated from femur and tibia in 15‐week‐old mice (male, n = 12 for WT, n = 12 for Gb3S KO). Data are expressed as mean ± S.D.
3. Discussion In this study, we demonstrated that deficiency of Gb3 synthase in mice attenuates bone formation. Among Gb3, Gb4, and Gb5, only Gb4 was expressed in osteoblasts. However, none of these were expressed in pre‐osteoclasts such as RAW264.7 and primary cultured pre‐osteoclasts derived from mouse bone marrow cells. Furthermore, 3D‐μCT, calcein double labeling, bone histomorphometric analyses, and expression analysis of osteogenic differentiation genes using Gb3S Int. J. Mol. Sci. 2019, 20, x 7 of 13
WT Gb3S KO 1.5 2 1.5
1.5 ) * 1 * 1 ) ) *
Alp 1 ( Runx2 ( 0.5
osteocalcin 0.5 0.5 ( Relative mRNA levels Relative mRNA levels Relative mRNA levels 0 0 0 Figure 7. Suppression of gene expression of osteogenic differentiation markers (Runx2, Alp, and osteocalcin) by Gb3 synthase deficiency. RNA isolated from femur and tibia in 15‐week‐old mice (male, n = 12 for WT, n = 12 for Gb3S KO). Data are expressed as mean ± S.D. The single asterisk indicates p < 0.05.
2.6. No Differences in Osteoclast Number and Expression of Osteoclast Differentiation Marker’s Genes (Nuclear Factor of Activated T‐Cells, Cytoplasmic 1 and Tartrate‐Resistant Acid Phosphatase) between WT and Gb3S KO Mice Bone resorption parameters such as osteoclast surface/bone surface (Oc.S/BS) and osteoclast numbers/bone surface (Oc.N/BS) were almost equivalent in the WT and Gb3S KO mice (Figure 8A,B). Furthermore, no differences in nuclear factor of activated T‐cells, cytoplasmic 1 (Nfatc1) and tartrate‐ Int.resistant J. Mol. Sci.acid2019 phosphatase, 20, 4619 (Trap) mRNA levels were found between WT and Gb3S KO mice (Figure7 of 13 8C).
WT Gb3S KO A B
30 15
20 10
10 5 Oc.S/BS(%)
0 Oc.N/BS(N/mm) 0
C 1.5 1.5
1 1 ） ） Trap
0.5 （ 0.5 Nfatc1 （ Relative mRNA levels 0 Relative mRNA levels 0 Figure 8. No eeffectsffects ofof Gb3Gb3 synthase synthase deficiency deficiency on on bone bone resorption resorption parameters parameters and and gene gene expression expression of osteoclastof osteoclast diff differentiationerentiation markers markers (Nfatc1 (Nfatc1and andTrap Trap). (A). )(A Osteoclast) Osteoclast surface surface/bone/bone surface surface (Oc.S (Oc.S/BS,/BS, %) in%) 15-week-old in 15‐week‐old mice mice (male, (male,n = n 9= for9 for WT, WT,n n= =10 10 for for Gb3S Gb3S KO). KO). ( B(B)) Osteoclast Osteoclast numbersnumbers/bone/bone surface (Oc.N(Oc.N/BS,/BS, numbernumber/mm)/mm) in 15-week-old15‐week‐old mice (male, n = 9 for WT, n = 10 for Gb3S KO). (C) mRNA expression ofof Nfatc1Nfatc1and andTrap Trap. RNA. RNA isolated isolated from from femur femur and and tibia tibia in 15-week-oldin 15‐week‐old mice mice (male, (male,n = n12 = for 12 WT,for WT,n = n12 = for 12 Gb3Sfor Gb3S KO). KO). Data Data are are expressed expressed as mean as meanS.D. ± S.D. ± 3. Discussion 3. Discussion In this study, we demonstrated that deficiency of Gb3 synthase in mice attenuates bone formation. In this study, we demonstrated that deficiency of Gb3 synthase in mice attenuates bone Among Gb3, Gb4, and Gb5, only Gb4 was expressed in osteoblasts. However, none of these were formation. Among Gb3, Gb4, and Gb5, only Gb4 was expressed in osteoblasts. However, none of expressed in pre-osteoclasts such as RAW264.7 and primary cultured pre-osteoclasts derived from these were expressed in pre‐osteoclasts such as RAW264.7 and primary cultured pre‐osteoclasts mouse bone marrow cells. Furthermore, 3D-µCT, calcein double labeling, bone histomorphometric derived from mouse bone marrow cells. Furthermore, 3D‐μCT, calcein double labeling, bone analyses, and expression analysis of osteogenic differentiation genes using Gb3S KO mice revealed histomorphometric analyses, and expression analysis of osteogenic differentiation genes using Gb3S that the deficiency of Gb3 synthase in mice decreased bone mass via attenuation of bone formation. It has been reported that among glycosphingolipids, gangliosides play an important role in the differentiation and proliferation of osteoblasts and osteoclasts. For instance, GD1a was reported to regulate differentiation from MSCs to osteoblasts via activation of epidermal growth factor receptor (EGFR) signaling [5,6]. GM1 also promotes differentiation from tendon stem cells to osteoblasts through attenuation of the phosphorylation of platelet-derived growth factor receptor-β (PDGFR-β)[8]. Furthermore, our recent study revealed that deficiency of GD3 synthase in mice results in the prevention of bone loss via the attenuation of bone resorption based on a decrease in osteoclast numbers [9]. This study revealed that genetic deletion of Gb3 synthase in mice decreased the number of osteoblasts and attenuated bone formation. Gb4 in osteoblasts may play a major role in this phenomenon, since osteoblasts did not express Gb3 and Gb5, but did express Gb4. This study also revealed that the expression of Gb4 in MC3T3 E1 cells was decreased after induction of osteoblastogenesis. We will need to examine which Gb4 is involved in the regulation of proliferation or differentiation in osteoblasts. To determine whether Gb4 in osteoblasts regulates the proliferation or differentiation, it is necessary to examine the effects of Gb4 treatment on osteoblasts derived from bone marrow or calvaria in Gb3S KO mice. Although there have been no reports that Gb4 regulates differentiation and proliferation of osteoblasts, it has been reported that this glycosphingolipid plays important roles in the regulation of cell signaling in various cells [18–22]. For instance, Gb4 promotes activation of extracellular signal-regulated kinases (ERK) signaling via association with EGFR [18] and is a potent inhibitor of protein kinase C (PKC) activity [19]. Gb4 also enhances the activity of transcription factors, Int. J. Mol. Sci. 2019, 20, 4619 8 of 13 activator protein-1 (AP-1) and cAMP response element binding protein (CREB) [20]. Furthermore, Gb4 upregulates Runx2 through TrkB-ERK signaling in dental epithelial cells [21]. Globo-series glycosphingolipids including Gb3 and Gb4, also increases the expression of P-glycoprotein through β-catenin signaling [22]. These signaling molecules such as ERK, PKC, AP-1, CREB, and β-catenin have been considered to be involved in proliferation and differentiation of osteoblasts [23–26]. Glycosphingolipids are enriched in glycolipid-enriched microdomain (GEM)/rafts on the plasma membrane of various cells [27], and enhance or attenuate the cell signals in the GEM/rafts [3,28–31]. Gb4 may also be localized in GEM/rafts, and interact with membrane molecules such as receptors, resulting in the formation of molecular complexes in the GEM/rafts, and promote the proliferation and/or differentiation of osteoblasts. Here, we propose the idea that Gb4 regulates the proliferation Int.and J./ Mol.or di Sci.fferentiation 2019, 20, x of osteoblasts in GEM/rafts as shown in Figure9. 9 of 13
FigureFigure 9. SchematicSchematic illustration illustration of of the the proposed proposed regulation regulation of of proliferation proliferation and/or and/or differentiation differentiation of of osteoblastsosteoblasts by by Gb4. Gb4.
4. MaterialsThe action and mechanismsMethods of globo-series glycosphingolipids in bone formation were not fully understood in this study. However, our observations provide the first evidence for the role 4.1.of globo-series Mice glycosphingolipids in bone formation. In summary, we have demonstrated that Gb4 is expressed in osteoblasts, and globo-series glycosphingolipids regulate bone mass through The generation of Gb3S KO mice was carried out as described previously [32]. WT and Gb3S KO bone formation. mice were mated, and the resultant heterozygotes were mated, and genotypes of the offspring were screened4. Materials as previously and Methods described [32]. The mice were housed under a 12 h light/dark cycle, and water and food were provided ad libitum. All protocols for animal experiments were approved by the Aichi Gakuin4.1. Mice University Animal Research Committee (approval number AGUD348; 24 May 2016) (Nagoya,The generationJapan), and of were Gb3S carried KO mice out was in accordance carried out with as described the National previously Institute [32 of]. WTHealth and Guide Gb3S KOfor themice Care were and mated, Use of and Laboratory the resultant Animals heterozygotes (1966). were mated, and genotypes of the offspring were screened as previously described [32]. The mice were housed under a 12 h light/dark cycle, and water 4.2. Cell Culture and food were provided ad libitum. All protocols for animal experiments were approved by the Aichi GakuinThe University MC3T3 E1 Animal mouse osteoblast Research Committee‐like cells, SaM (approval‐1 human number osteoblast AGUD348; cells [33], 24 May RAW264.7 2016) (Nagoya, mouse preJapan),‐osteoclast and were (monocyte/macrophage) carried out in accordance cells, with and themouse National bone marrow Institute cells of Health isolated Guide from for long the bones Care (femurand Use and of Laboratorytibia) [34] were Animals cultured (1966). in α‐Minimum Essential Media with 10% fetal bovine serum and antibiotics (100 U/mL penicillin, 100 μg/mL streptomycin; Wako Pure Chemical Industries, Ltd., Osaka,4.2. Cell Japan). Culture Cells were maintained at 37 °C with 5% CO2 in a humidified incubator. The MC3T3 E1 mouse osteoblast-like cells, SaM-1 human osteoblast cells [33], RAW264.7 mouse 4.3.pre-osteoclast Antibodies (monocyte/macrophage) cells, and mouse bone marrow cells isolated from long bones (femurAnti and‐Gb3 tibia) monoclonal [34] were culturedantibody in (mAb)α-Minimum c61 was Essential generated Media in Dr. with Furukawa’s 10% fetal bovine laboratory serum [35]. and Anti‐Gb4 mAb HIRO34 was as described in [36]. Anti‐Gb5 (SSEA‐3) mAb and FITC‐conjugated anti‐ mouse IgM were purchased from Affymetrix eBioscience (San Diego, CA, USA). FITC anti‐human IgM and anti‐rat IgM were purchased from BioLegend (San Diego, CA, USA).
4.4. Induction to Mature Osteoblasts The MC3T3 E1 cells were plated in 60 mm dishes. When cells were confluent, 50 μg/mL of ascorbic acid (Wako Pure Chemical Industries, Ltd.) and 5 mM β‐glycerophosphate (Sigma‐Aldrich, St. Louis, MO, USA) were added. The medium was changed every other day. After 7 and 21 days of culture, cells were used for flow cytometry or quantitative real‐time PCR.
4.5. In Vitro Osteoclast Induction Mouse bone marrow cells were plated in 150 mm dishes and cultured with 10 ng/mL macrophage colony‐stimulating factor (M‐CSF; PeproTech, Inc., Rocky Hill, NJ, USA) for 3 days. The Int. J. Mol. Sci. 2019, 20, 4619 9 of 13 antibiotics (100 U/mL penicillin, 100 µg/mL streptomycin; Wako Pure Chemical Industries, Ltd., Osaka, Japan). Cells were maintained at 37 ◦C with 5% CO2 in a humidified incubator.
4.3. Antibodies Anti-Gb3 monoclonal antibody (mAb) c61 was generated in Dr. Furukawa’s laboratory [35]. Anti-Gb4 mAb HIRO34 was as described in [36]. Anti-Gb5 (SSEA-3) mAb and FITC-conjugated anti-mouse IgM were purchased from Affymetrix eBioscience (San Diego, CA, USA). FITC anti-human IgM and anti-rat IgM were purchased from BioLegend (San Diego, CA, USA).
4.4. Induction to Mature Osteoblasts The MC3T3 E1 cells were plated in 60 mm dishes. When cells were confluent, 50 µg/mL of ascorbic acid (Wako Pure Chemical Industries, Ltd.) and 5 mM β-glycerophosphate (Sigma-Aldrich, St. Louis, MO, USA) were added. The medium was changed every other day. After 7 and 21 days of culture, cells were used for flow cytometry or quantitative real-time PCR.
4.5. In Vitro Osteoclast Induction Mouse bone marrow cells were plated in 150 mm dishes and cultured with 10 ng/mL macrophage colony-stimulating factor (M-CSF; PeproTech, Inc., Rocky Hill, NJ, USA) for 3 days. The surface-attached cells were used as osteoclast precursors [34]. These precursors were cultured with 10 ng/mL M-CSF and 50 ng/mL RANKL (PeproTech, Inc.). After 24 h of treatment with RANKL, the cells were used for flow cytometry. RAW264.7 cells were plated in 150 mm dishes and cultured with 50 ng/mL RANKL. After 2, 3, and 4 days of treatment with RANKL, the cells were used for flow cytometry or quantitative real-time PCR.
4.6. Flow Cytometry Cell surface expression of Gb3, Gb4, and Gb5 was analyzed by AccuriTM C6 Flow Cytometer (BD Biosciences, San Jose, CA, USA) using anti-Gb3 (mouse IgM, c61), Gb4 (human IgM, HIRO34), and Gb5 (rat IgM, MC-631) mAbs. The cells were incubated with individual antibodies for 1 h on ice and then stained with FITC-conjugated anti-mouse, human, or rat IgM for 45 min on ice. Control cells for flow cytometry were prepared using the secondary antibody alone.
4.7. Quantitative Real-Time PCR (qPCR) Total RNA was extracted using a RNeasy Plus mini kit (Qiagen, Germantown, MD, USA). Reverse transcription was conducted with a high-capacity cDNA reverse transcription kit (Applied Biosystems, Carlsbad, CA, USA), and quantitative real-time PCR was performed using TaKaRa Thermal Cycler Dice Real Time System III with THUNDERBIRD SYBR qPCR mix kits (TOYOBO, Osaka, Japan). Total RNA was used for reverse transcription and the cycling conditions were 25◦C for 10 min, 37◦C for 120 min, and 85◦C for 5 min. The PCR cycling conditions were 95◦C for 10 min (pre-denaturation), 40 cycles at 95◦C for 15 sec (denaturation), and 60◦C for 1 min (extension) [9]. We evaluated the mRNA levels of A4galt, B3galnt1, Runx2, Alp (alkaline phosphatase), osteocalcin, Nfatc1, and Trap using primers listed in Table1. Gapdh was used for an internal control. Int. J. Mol. Sci. 2019, 20, 4619 10 of 13
Table 1. Real-time PCR primers used in this study.
Target Forward Primer Backward Primer
A4galt 50-CCTGTTCCCATCTGGAGGAG-30 50-CCCTTTCATCAGCACAACCA-30
B3galnt1 50-TCTTGACTGCCCTTCCCAAT-30 50-GGAGCGTGAAGCGAAAGTCT-30
Runx2 50-CCCAGCCACCTTTACCTACA-30 50-TATGGAGTGCTGCTGGTCTG-30
Alp 50-AACCCAGACACAAGCATTCC-30 50-GCCTTTGAGGTTTTTGGTCA-30
osteocalcin 50-CCGGGAGCAGTGTGAGCTTA-30 50-AGGCGGTCTTCAAGCCATACT-30
Nfatc1 50-GGTGCTGTCTGGCCATAACT-30 50-GCGGAAAGGTGGTATCTCAA-30
Trap 50-TCCTGGCTCAAAAAGCAGTT-30 50-ACATAGCCCACACCGTTCTC-30
Gapdh 50-TGCACCACCAACTGCTTAG-30 50-GGATGCAGGGATGATGTTC-30
4.8. Three-Dimensional Micro-Computed Tomography (3D-µCT Analysis) The distal region of the femur was scanned using 3D-µCT (CosmoScan R-mCT-GX-T1; RIGAKU, Tokyo, Japan), and the parameters of cancellous bone were analyzed by TRI/3D-BON (Ratoc, Tokyo, Japan) software as described previously [9]. In brief, the scanning was initiated at 0.5 mm above the distal femoral growth plate, and a total of 75 consecutive 20-µm-thick sections were analyzed.
4.9. Measurement of Bone Formation Rate Using Calcein Double Labeling Calcein double labeling was performed to calculate mineral surface/bone surface (MS/BS), mineral apposition rates (MAR), and bone formation rate (BFR). We injected mice with calcein (8 µg/g; Sigma, St. Louis, MO, USA) intraperitoneally at 3 days and 1 day before euthanasia. The femurs were fixed with 4% paraformaldehyde and sectioned at 5 µm thickness as undecalcified sections. To measure MS/BS, MAR, and BFR, we used the metaphyseal cancellous bone in the femur to obtain the bone fraction in a rectangular area of 0.34 mm2 (0.5 mm 0.67 mm), with its closest and furthest edges being × 0.5 and 1.0 mm medial to the growth plate, respectively [37].
4.10. TRAP Staining TRAP staining was conducted as described previously [38]. In brief, the slides (femur samples) were incubated in sodium acetate buffer (0.1 M, pH 5.0) containing naphthol AS-MX phosphate, Fast Red Violet LB Salt, and MnCl2 in the presence of sodium tartrate at 37◦C for 60 min for TRAP staining.
4.11. Bone Histomorphometric Analyses Femur samples were fixed with 4% paraformaldehyde and decalcified in 10% ethylenediaminetetraacetic acid (EDTA) for three weeks, embedded in paraffin, and sectioned at 5 µm thickness. These slides were used for hematoxylin and eosin (HE) and TRAP staining. After staining with HE, osteoblast numbers on the bone surface (Ob.N/BS) were evaluated as described previously [9]. Osteoclasts were stained with TRAP, and osteoclast surface on the bone surface (Oc.S/BS) and osteoclast numbers on the bone surface (Oc.N/BS) were evaluated by scoring the TRAP-positive cells on the bone surface [9]. The parameters were measured within an area of 0.8 mm2 (1.0 mm 0.8 mm), with the × closest and furthest edges being 2.0 and 3.0 mm medial to the growth plate of the proximal ends of the femur, respectively [39].
4.12. Statistical Analysis All data were expressed as mean S.D. Statistical significance was evaluated using a Student’s ± t-test at p < 0.05 and the single and double asterisks indicate p < 0.05 and p < 0.01, respectively. Int. J. Mol. Sci. 2019, 20, 4619 11 of 13
Author Contributions: K.H. (Kazunori Hamamura), K.F., K.M., S.G., and A.T. designed the research. K.H. (Kazunori Hamamura), H.K., and K.T. contributed to the methodology. K.H. (Kazunori Hamamura), K.H. (Kosuke Hamajima), S.Y., Y.M., and H.M. performed the experiments and analyzed the data. M.U. and Y.K. provided anti-Gb4 and Gb3 mAbs, respectively. K.H. (Kazunori Hamamura) wrote the manuscript. K.H. (Kazunori Hamamura) acquired funding. All authors read and approved the final manuscript. Funding: This work was supported by JSPS KAKENHI Grant Number JP17K11657 and by a Research Grant from the Center for Advanced Oral Science, Aichi Gakuin University to KaH. Acknowledgments: The authors are grateful to Ohmi. Y, Furukawa Keiko, and Nakayasu. Y for technical assistance. Conflicts of Interest: The authors declare no conflict of interest.
References
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